With the completion of the Human Genome Program approaching, there is an increasing interest in studying the function of genes, particularly those involved in human development and disease. While mapping and nucleotide sequencing of genes is an important first step for understanding the function of genes, the physical characterization of the structure of a gene does not provide insight into the function of that gene in the context of a multicellular organism.
For example, prior art approaches to determining gene function in mammals have relied on targeting mutations to specific genes in embryonic stem (ES) cells, or on genome-wide mutagenesis techniques designed to mutate all genes of an organism (e.g., mice). For example, “knock-out” mutations in ES cells have been widely used to target mutations to specific genes. “Knock-out” mutations shut off or alter gene expression and are currently used to produce a phenotype in the whole animal which reflects the function of the knocked-out gene. This approach has identified many genes which are associated with cancer and other human genetic diseases, and relies either on phenotype-based screens (i.e., screening for a particular phenotype) or on gene-based screens (i.e., screening for a particular alteration in the genome).
Phenotype-based screens have primarily been conducted using mice, and involve characterization of thousands of mutagenized mice for specific diseases and traits [Russell et al., Proc. Natl. Acad. Sci. USA 76:5818-5819, 1979; Hitotsumachi et al., Proc. Natl. Acad. Sci. USA 82:6619-6621; Shedlovsky et al., Genetics 134:1205-1210; Marker et al., Genetics 145:435-443, 1997]. While the phenotype-based approach has the advantage that no assumption is made with respect to which genes are associated with a given disease or disorder, it is nevertheless very costly when using organisms such as mice since it requires the maintenance of several lines of mutagenized whole organisms. Furthermore, it is unclear whether phenotype-based screens permit conducting saturation screens for both dominant and recessive mutations of all mouse genes.
Gene-based screens have been carried out in whole animals and in embryonic stem (ES) cells. This approach involves identifying the organism's genes or the ES cell genes which have been mutated. Homologous recombination and retroviral insertion are commonly used in ES cells [Zambrowicz et al. (1998) Nature 392:608-611]. Although mutagenesis by homologous recombination is becoming routine, it remains cumbersome and expensive. Similarly, while the genome-wide approach to mutagenizing ES cells by retroviral insertional mutagenesis allows the generation of a large number of mutagenized ES cells in a cost effective manner, this approach produces only one, or a limited number of, alleles of a given gene. Additionally, the class of mutations that can be produced with this approach is limited to those mutations which result from integration of a retroviral element. Thus, mutations caused by, for example, single amino acid changes in the protein cannot be produced using this approach. In many instances, for example, it may be desirable to generate mutations which cause single amino acid changes that merely modify gene function (e.g., by generating hypomorphic alleles that express the gene with a reduced efficiency) or that give rise to a new trait in the animal (e.g., by generating dominant neomorphic alleles which result in a gain of function). The generation of hypomorphic and neomorphic alleles of a gene in a model organism by single amino acid substitutions may be desirable to create a model organism for a human trait or disease in which gene function is modified rather than destroyed.
Accordingly, what is needed are methods for determining gene function which may efficiently be applied on a genome-wide scale, which generate more than one mutation in a gene of interest, and which do not only abrogate the function of the gene.